1. Field of the Invention
This invention relates to derivatives of human-originated non-immunogenic plasminogen activators, and more particularly to such a derivative comprising at least one polyalkylene glycol attached with at least one coupling agent to amino acid side chains of a plasminogen activator of the type described above, the polyalkylene glycol having a molecular weight in the range of 200-20,000 and optionally containing one or more alkyl, alkoxy and/or alkanoyl groups as substituents. Further, the invention is concerned with a process for producing such derivatives and with a thrombolytic agent containing such derivatives.
2. Description of the Prior Art
It is known that human tissues contain a variety of substances which activate plasminogen into a fibrinolytic enzyme or plasmin. Among such known substances, the most representative is a plasminogen activator, i.e. urokinase, which is formed in the kidney tissue and excreted into urine. Urokinase may be obtained by isolation and purification from human urine, tissue culture or genetic engineering. As fibrinolytic enzyme activators which have nowadays found widespread commercial utility, there exist proteins originated from hemolytic Streptococcus and urokinase which is an enzyme originated from human urine. In view of its immugenic behavior towards humans, urokinase resulting from such source is favorably employed for clinical application. Urokinase originated from human urine is believed to contain both high molecular weight urokinase (molecular weight: 54,000) and low molecular weight urokinase (molecular weight: 33,000). Urokinase has been used, in recent years, as a thrombolytic agent or an adjuvant for carcinostatic substances, and its consumption for clinical application is increasing year by year.
However, urokinase is unstable under certain conditions since it is an enzyme and loses its enzymatic activity, for example, in the course of extraction, isolation and purification from a urokinase-bearing raw material, for example, urine; during the lyophilization processing in preparing dosable formulations; during the heat treatment for deactivating viruses; or when it is placed in a diluted state in a dripping bottle and kept for a prolonged time period in such a diluted state at room temperature for clinical application. This physically unstable nature of urokinase has created a serious problem in preparing and formulating urokinase on an industrial scale or in actually using the same for clinical purposes. Human albumin has been employed as an additive to urokinase so as to improve its stability. However, this can be by no means a break-through solution to the problem just discussed because pure albumin, i.e. a globulin fraction, is difficult to obtain without immunogenic contamination; pure albumin is expensive; albumin and urokinase form a complex of a high molecular weight under virus deactivating conditions in which urokinase is subjected to heat treatment at 60.degree. C. for 10 hours together with albumin added to stabilize urokinase; and such stabilizer if added may be effective to a certain extent for protecting urokinase from losing its enzymatic activity upon the lyophilization but cannot prevent its loss of activity upon actual clinical use.
The physiological activity of urokinase when administered intravenously to living bodies is promptly retarded by protease inhibitors present in blood (.alpha..sub.2 macroglobulin, and .alpha..sub.2 -plasmin inhibitors and the like), and the metabolic rate of urokinase per se is very high, resulting in extremely shortened half-life which does not exceed even several minutes. Nothing has been heretofore proposed to solve the problem of short half-life of urokinase in blood.
The present inventors have carried out extensive research with a view toward developing derivatives of human-originated non-immunogenic plasminogen activators which will overcome the above-noted drawbacks of the prior art techniques. As a result, they have succeeded in finding plasminogen activator derivatives which are stable and hardly retarded by inhibitors present in blood and hence achieve prolonged half-life in blood, thereby leading to the present discovery.